Various 2-arylbenzazole compounds found to be active in inhibiting proliferation of certain tumor cells and exemplified by 2-(4'-aminophenyl) benzothiazole and close analogues or acid addition salts thereof are disclosed in PCT international patent application No. PCT/GB94/01883 published Mar. 9, 1995 under No. WO 95/06469.
The compounds with which the present invention is concerned are also 2-arylbenzazole compounds which are believed to comprise novel or new chemical entities and which are of particular interest as active chemotherapeutic agents for use in therapy, especially antitumor therapy, by virtue of an ability to inhibit proliferation of certain tumor cells.
For some of the benzazole compounds disclosed in the aforesaid PCT international patent application, for instance the compound 2-(4'-aminophenyl) benzothiazole which has been designated the reference code CJM 126, a remarkably high specific inhibitory activity has been found in respect of certain human breast cancer cell lines. It has now also been found, however, that some of the compounds previously disclosed in said prior PCT application, and benzazole compounds newly disclosed in the present application, can exhibit anti-proliferative activity selectively in respect of a number of different cell lines that relate to a range of various mammalian cancers other than human breast cancer. The present invention accordingly envisages the use of 2-arylbenzazole compounds as specified for making medicaments or pharmaceutical compositions for use in antitumor therapy not necessarily only for the treatment of breast cancer but additionally, or alternatively, for the treatment of certain other selected cancers.
More specifically, the benzazole compounds of the present invention are generally 2-arylbenzazole compounds represented by the structural formula I below, or a pharmaceutically acceptable salt thereof, ##STR1## characterised in that X is S or O;
R.sup.1 and R.sup.3 are each independently hydrogen, alkyl, hydroxyl, alkoxy, or aralkoxy; PA1 R.sup.2 is selected from hydrogen, NO.sub.2, NH.sub.2, halogen, alkyl, CN, and a substituted alkyl oxysulphonyl group; PA1 R.sup.5 and R.sup.6 are each independently hydrogen, alkyl, or an acyl or benzoyl group ##STR2## where Y is O or S, and R.sup.8 is alkyl (including cyclo-alkyl), a halogenated lower alkyl, or phenyl, PA1 where M.sup.+ is a monovalent cation or cationic group, and PA1 R.sup.7 is hydrogen, 5'-halogen or 5'-alkyl PA1 (a) when R.sup.5 and R.sup.6 are each hydrogen or alkyl, R.sup.2 is not hydrogen but is a 3'-substituent in the phenyl group other than a 3'-substituted alkyl oxysulphonyl group; PA1 (b) R.sup.7 is limited to being hydrogen unless R.sup.2 is a 3'-substituent in the phenyl group; PA1 (c) if R.sup.2 is NO.sub.2 it is a 3'-substituent in the phenyl group; PA1 (d) alkyl groups when present as such in the compound or as a moiety in other groups such as alkoxy are each composed of less than 6 carbon atoms; PA1 (e) the compound is not 2-(4'-amino-3'-iodophenyl) benzothiazole (unless in the form of a sulphamate salt thereof). PA1 (a) at least some alkyl groups when present as such or as a moiety in other groups such as alkoxy are methyl or ethyl; PA1 (b) halogen substituents, when present, are selected from iodine, bromine and chlorine. PA1 or a halogenated lower alkyl, PA1 or phenyl. PA1 Cells were maintained in a continuous logarithmic culture in Dublecco's medium supplemented with 10% fetal calf serum and penicillin (100 IU/ml) and streptomycin (100 .mu.m). The cells were mildly trypsinized for passage and for use in assays. On day zero, 100 .mu.l of trypsinized tumor cells (1.times.10.sup.4 /ml) were plated in the wells of 96-well flat-bottom microtiter plates. The plates were incubated for 2 days at 37.degree. C. and 5% CO.sub.2 in air to allow the cells to adhere and resume exponential growth prior to the addition of drugs. PA1 On day 7 the test was terminated by the addition of 100 .mu.l saline containing 0.002% w/v propidium iodide (Sigma), 0.3% drawing ink (Staedtler "Marsmatic 745"--Trade Mark) and 0.5% Triton X-100. The plates were kept at 4.degree. C. overnight before reading on an inverted microscope equipped with an automated scanning stage. Fluorescence intensity was measured in arbitrary units by a photomultiplier. An HP-87 computer controlled the movement of the stage and also collected and processed the data from the multiplier. PA1 For each compound tested a dose-response curve was obtained and the IC.sub.50 value (the drug concentration at 50% inhibition of cell growth) was calculated. PA1 Curve A represents a saline control; PA1 Curve B shows the results for a dose of 6.25 mg/Kg administered by injection; PA1 Curve C shows the results for a dose of 12.5 mg/Kg administered by injection; PA1 Curve D shows the results for a dose of 25 mg/Kg administered by injection. PA1 A mixture of the 2-(nitrophenyl) benzazole compound in question (0.05 Mol,) and stannous chloride dihydrate (0.25 Mol.) in absolute ethanol (200 ml) is stirred and refluxed under nitrogen for 1 to 4 hours. The ethanol is then removed under reduced pressure and the residue is dissolved in ethyl acetate (4.times.100 ml). The combined organic phases are next shaken with excess aqueous sodium hydroxide to liberate the free amine bases and dissolve the tin residues. The separated organic phase is washed with water, dried (magnesium sulphate) and the solvent is evaporated. Finally, the products are then crystallised.
or SO.sub.3.sup.- M.sup.+
subject to the following provisos:
Preferred compounds of the invention in accordance with formula I wherein R.sup.3 is hydrogen include compounds in which R.sup.1 is alkyl, alkoxy or benzyloxy. It is also usually preferred that X be sulfur. Preferred compounds of the invention in accordance with the structural formula I may also be further characterised by at least one of the following features:
It has been found that at least for compounds of structural formula I wherein R.sup.5 and R.sup.6 are both hydrogen, i.e. wherein the phenyl group has a 4'--NH.sub.2 substituent, a very effective degree of anti-proliferative activity against various mammalian tumor cells may arise when R.sup.2 is a halogen atom, or is a lower alkyl group (preferably Me or Et), in the 3' position of the phenyl group. For example, the particular combinations of 4'--NH.sub.2 and 3'--Cl, 4'--NH.sub.2 and 3'--Br, 4'--NH.sub.2 and 3'--I, 4'--NH.sub.2 and 3'--Me, and 4'--NH.sub.2 and 3'--Et in the phenyl group of the 2-aryl component have been found to yield compounds with potent anti-proliferative properties against at least some selected tumor cells. The 3' position substituent may alternatively be a cyano group, giving a further combination 4'-NH.sub.2 and 3'--CN.
In these compounds in which R.sup.2 is a 3'-substituent in the phenyl group, when R.sup.1 is an alkyl, alkoxy or benzyloxy substituent it is generally preferred that R.sup.1 should be a substituent in the 6-position of the benzazole moiety.
Compounds in accordance with the invention which conform to formula I wherein R.sup.2 is a 3'-substituent in the phenyl group, and which are of particular interest, include those compounds where R.sup.5 and R.sup.6 are both hydrogen and the combination of substituents R.sup.1, R.sup.2, R.sup.3, R.sup.7 and X is selected from the following combinations:
______________________________________ R.sup.1 R.sup.3 X R.sup.2 R.sup.7 Ref. No. ______________________________________ H H S 3'-Me H (DF203) H H S 3'-Et H (DF223) 6-Me H S 3'-I H (DF219) 6-OMe H S 3'-I H (DF210) H H O 3'-1 H (DF206) H H S 3'-Br H (DF209) 6-Me H S 3'-Br H (DF220) H H S 3'-Cl H (DF229) H H S 3'-CN H (DF230) H H S 3'-Br 5'-Br (126) H H S 3'-Cl 3'-Cl H H S 3'-Cl 5'-Me ______________________________________
Another group of benzazole compounds which provide some very promising anti-proliferative agents for use in antitumor therapy are compounds conforming to structural formula I wherein the substituent NR.sup.5 R.sup.6 is an N-acyl or N-diacyl derivative) or equivalent benzoyl derivative) e.g. ##STR3## where, as hereinbefore specified, Y is O or S and R.sup.8 is a lower alkyl (including a cyclised lower alkly such as cyclobutyl),
Acyl or benzoyl derivatives as referred to above which are of particular interest include those compounds where NR.sup.5 R.sup.6 is an N-acyl group (or N-benzoyl group) and where the combination of substituents R.sup.1, R.sup.2, R.sup.3, R.sup.8, X and Y is selected from the following combinations.
______________________________________ R.sup.1 R.sup.3 X R.sup.2 Y R.sup.8 Ref. No. ______________________________________ H H S H O Me (DF128) H H O H O Me (DF140a) H H S H S Me (DF188) H H O H S Me (DF175) H H S H O CH.sub.2 Cl (DF180) H H O H O CH.sub.2 Cl (DF190) H H O 3'-I O CH.sub.2 Cl (DF225) H H O 3'-NO.sub.2 O Me (DF214) H H S H O CHCl.sub.2 (DF232) H H S H O Ph (DF131) H H S H O Cyclobutyl (KF497) ______________________________________
Reference code numbers are denoted in brackets for some of the above compounds for which more detailed preparative examples are hereinafter presented.
It will also be understood that many of the compounds in accordance with the invention which are herein referred to may be presented in the form of pharmaceutically acceptable salts, especially acid addition salts derived from an acid selected for example from the group comprising: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, salicyclic, p-toluenesulphonic, tartaric, citric, lactobionic, formic, malonic, pantothenic, succinic, naphthalene-2-sulphonic, benzene-sulphonic, methanesulphonic and ethanesulphonic.
It should also be understood, however, that where reference is made in this specification to compounds of formula I such reference should be construed as extending not only to their pharmaceutically acceptable salts but also to other pharmaceutically accepatble bioprecursors (pro-drug forms) where relevant. Moreover, where any of the compounds referred to can exist in more than one enantiomeric form, all such forms, mixtures thereof, and their preperation and uses are within the scope of the invention.
More particularly, sulphamate salts consititing potential water-soluble pro-drug forms of the 2-(aminophenyl) benzazole compounds previously mentioned, especially para amino or 4'--NH.sub.2 derivatives, provide a further category of promising benzazole compounds within the scope of the present invention. These sulphamate salts may break down in biological systems to form corresponding amines, and will generally be compounds conforming to structure I wherein NR.sup.5 R.sup.6 is 4--NHSO.sub.3.sup.- M.sup.+ as hereinbefore defined. In preferred embodiments M.sup.+ is an alkali metal cation such as Na.sup.+ or is a cationic group such as NH.sub.4.sup.+.
Like acyl derivatives such as N-acetyl and N-chloro-acetyl derivatives, and like other acid addition salts, e.g. hydrochloride, dihydrochloride, methanesulphonic acid and ethanesulphonic acid addition salts, these sulphamate salts are expected to be equally effective in inhibiting proliferation of tumor cells in antitumor therapy as the parent amino compounds from which they may be considered to be derived. The salts may of course dissociate in water or other aqueous media to provide the active antitumor compound, and in practice these water soluble compounds are likely to be the most preferred compounds for making up acceptable pharmaceutical formulations. It may for example be noted that the sulphamate salt hereinafter described and designated by the reference code DF183 has been found to have an aqueous solubility of about 10 mg/ml whereas that of the compound 2-(4'-aminophenyl) benzolthazole referred to as CJM 126 has an aqueous solubility of only 3.8 .mu.g/ml.
Specific sulphamate salts in accordance with formula I which are of particular interest include compounds in which the combination of substituents R.sup.1, R.sup.2, R.sup.3, NR.sup.5 R.sup.6 and X is selected from the following combinations:
______________________________________ R.sup.1 R.sup.3 X R.sup.2 NR.sup.5 R.sup.6 Ref. No. ______________________________________ H H S H NHSO.sub.3.sup.- Na.sup.+ DF183 H H S H NHSO.sub.3.sup.- NH.sub.4.sup.+ DF191 H H O H NHSO.sub.3.sup.- Na.sup.+ DF187 H H S 3-I NHSO.sub.3.sup.- Na.sup.+ DF224 H H S 3-Me NHSO.sub.3.sup.- Na.sup.+ DF228 ______________________________________
The invention also comprises the use of a 2-arylbenzazole compound as specified above for therapy, especially for making a medicament or pharmaceutical composition for selective use in antitumor therapy.
As hereinafter described, the invention also includes pharmaceutical compositions or preparations, conveniently in unit dosage form, for selective use in antitumor therapy, said compositions or preparations comprising as the active substance a 2-arylbenzazole compound as herein specified.